DESCRIPTION: (Applicant's Abstract): Project 2 (Rapid Gene Discovery) has as its major goal the discovery of the gene(s) disrupted by or in the vicinity of individual balanced chromosome aberrations associated with developmental abnormalities. We will receive BAC clones from Project 1 (High Throughput FISH Mapping) that cross the sites of balanced aberrations and DNA from the corresponding patients. Breakpoints will be prioritized for analysis to maximize the likelihood that the break is responsible for the patient phenotype or that genes near the break can be effectively assessed as candidates. Using PCR-based strategies from the human genomic sequence, we will collaborate with Project 1 on detection of the site of the chromosomal breakpoint within the BAC. The sequence of altered chromosomes will be compared with the corresponding normal sequences to determine the extent of any deletion, duplication or other rearrangement at the breakpoints. We will identify genes in the vicinity of the breakpoint on each chromosome by a combination of database searching, cross-species comparison, computation gene prediction, RT-PCR and PCR-based and hybridization-based cDNA library screening. Genes disrupted by the breakpoint will be identified by cDNA FISH (Project 1) and by probing Southern blots of patient DNA with cDNAs. We will use Northern blot and RT-PCR analysis to assess whether the disrupted genes produce novel transcripts and will extend selected cDNAs to define the full coding sequence, screening these for protein motifs or homologies that might predict gene function. Where possible, disrupted genes or candidates for disregulation by position effects will be analyzed for mutations in an independent set of non-translocation patients displaying similar phenotypes. Our goal is to initially analyze 8 individual breakpoints per year, drawn from the chromosomes for which Project 1 identifies "crossing" BACs either directly, or through the help of this project for genomic walking in GenBank or a BAC library. Genes discovered in Project 2 will be forwarded to Project 3 for analysis of developmental expression and prioritization based on all available data of the most compelling choices for functional genetic analysis in model systems.